Articular cartilage is composed of chondrocytes encased within the complex extracellular matrix produced by these cells. The unique biochemical composition of this matrix provides for the smooth, nearly frictionless motion of articulating surfaces of the knee joint. With age, tensile properties of human articular cartilage change as a result of biochemical changes. After the third decade of life, the tensile strength of articular cartilage decreases markedly. Damage of cartilage caused by trauma or disease, e.g., rheumatoid and osteoarthritis, can lead to serious physical debilitation. The repair of defective cartilages has been improved recently by transplantation of autogenous or allogenous chondrocytes, or through the use of mesenchymal stem cells.1-4 Moreover, U.S. Pat. No. 6,623,963 discloses a biocompatible, resorbable Type II collagen-based matrix is reconstituted from solubilized animal cartilage tissue and used in the culture and growth of cells, such as chondrocyte cells. However, the limited source for sufficient amount of normal tissues or cells restricts the development and clinical application of these cell-based therapies. Moreover, during in vitro monolayer expansion of harvested chondrocytes, the cells are eventually lose their phenotypic features, such as attenuation of type II collagen production and reduction in proteoglycan accumulation.5,6 
These cellular alterations are probably resulted from changes in chondrocyte functions or the reduced ability of these cells to maintain normal synthetic activity during serial monolayer culture. Less uniform aggrecan and functional proteins, and altered responsiveness to anabolic growth factors, such as transforming growth factor β1 (TGF-β1) and insulin-like growth factor I (IGF-I)7-9 were observed in these cells. The collagen typing of the progeny has been shown to shift from type II to type I collagen during the rapid proliferation to quiescence.5,6 Almost invariably, the serially expanded chondrocytes do not retain their original round or polygonal appearance, but gradually become fibroblast-like cells, and then fully extended and flattened on the substratum during monolayer expansion. This kind of chondrocytes was referred as ‘dedifferentiated’ since it no longer possesses the general phenotypic features and functions of freshly harvested chondrocytes.5,6 Numerous factors, including the seeding density, culture medium and age of the cell source, affect the extent and rate of this process.10,11 Loss of chondrocyte phenotype and functions during serial expansion in vitro poses a key limitation to the commercialization and/or clinical use of the orthobiologic approaches of cartilage repair.
The two major extracellular components of articular cartilage are the aggregated proteoglycan consisting aggrecan, and type II collagen. The type II collagen forms the backbone of cartilage, providing it with stability and tensile strength. The highly hydrated proteoglycan component is responsible for the compressive stiffness of cartilage. In addition to playing an important structural framework, the extracellular matrix (ECM) component has also been shown to contribute to the regulation of chondrocytes through cell-surface signaling mechanisms.15-17 These complex interactions would influence chondrocyte gene expression at mRNA levels as well as protein levels, thereby changing the internal status of the cell through its display of surface receptors and ECM molecules.17 Matrix deterioration often associated with the pathological state of the cartilage, in which type II collagen is reduced and activity of metalloproteinases increased.8,9 Furthermore, TGF-β1 and IGF-I have been implicated as important mediators in the induction and maintenance of matrix macromolecules in articular cartilage.4,18-21 
Loss of chondrocyte phenotype during serial expansion in vitro poses a key limitation to the commercialization and/or clinical use of orthobiologic approaches to articular cartilage repair. To counter dedifferentiation, chondrocytes traditionally have been suspended in three-dimensional environments such as hydrogels, e.g., agarose or alginate, pellet culture, or three-dimensional scaffolds. Several approaches for chondrocyte redifferentiation have been developed. Some studies demonstrated that ‘dedifferentiated’ chondrocytes could be ‘redifferentiated’ while cultured three-dimensionally in gels of agarose, collagen or alginate.8,12-14 However, using this method, only the chondrocytes subcultured up to passage 4 (P4) can be restored to express cartilaginous phenotype. Accordingly, the numbers of expanded chondrocytes which are still retaining the ability to restore native chondrocyte phenotype can only be achieved at about 23.8 folds of expansion of the original cell number, which are insufficient for commercial and/or clinical use. Furthermore, the procedure for three-dimensional culture in alginate bead is complex and may take at least two weeks to induce restoration of cartilaginous phenotype. Moreover, the chondrocytes need to be isolated from alginate beads for further applications, such as autogenous or allogenous transplantation. All the above disadvantages make the three-dimensional methods ineffective.
U.S. Pat. No. 7,273,756 discloses a method of maintaining chondrocyte phenotype during serial expansion by culturing a population of chondrocytes in a defined serum-free culture medium containing cytokines and on a substrate that is modified by covalent attachment of hyaluronic acid. U.S. Pat. No. 7,189,567 provides a method for rapidly culturing a large amount of human chondrocytes to give normal chondrocytes or a mass thereof, comprising co-culturing human chondrocytes together with perichondral cells in the chondrogenic stage, as feeder cells, which support the proliferation ability of the chondrocytes, to allow rapid culturing of the human chondrocytes in a large amount. Yaeger P. C. et al. demonstrated that synergistic action of TGF-β1 and IGF-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes.30 However, addition of cytokines, hyaluronic acid or growth factors for restoration of chondrocyte phenotype in chondrocyte cell culture is cost ineffective. Furthermore, only the chondrocytes subcultured up to passage 4 (P4) can be restored to express cartilaginous phenotype, and the cells of the subsequent passages cannot be redifferentiated. Accordingly, the numbers of redifferentiated chondrocytes can only achieve about 23.8 folds of the original number, which is insufficient for commercial and/or clinical use.
In view of the above, there exists a need for a convenient and effective method for restoring phenotypic expression of expanded chondrocytes.